A hybridoma cell line 10g4 and a monoclonal antibody against the total of aflatoxin b1, b2, g1 and g2

ABSTRACT

Hybridoma cell line 10G4 and monoclonal antibody against total aflatoxins produced by the hybridoma cell line 10G4. The hybridoma cell line 10G4 is used to produce the monoclonal antibody that binds specifically total aflatoxin B1, B2, G1 and G2. The titer of the mouse ascites antibody produced by the 10G4 treated mouse is determined through non-competitive enzyme-linked immunosorbent assay and the titer can reach up to 5.12×10 5 . The monoclonal antibody against total aflatoxin B1, B2, G1 and G2 are used for better identification of aflatoxin B1, B2, G1 and G2 with good consistency. The 50% inhibitory concentrations (IC 50 ) of the antibody against aflatoxin B1, B2, G1 and G2 are 2.09 ng/mL, 2.23 ng/mL, 2.19 ng/mL and 3.21 ng/mL respectively. The range of cross reaction rate for aflatoxin B1, B2, G1 and G2 is about 65.2%-100%. The antibody is used for quantitative measurement of total aflatoxin B1, B2, G1 and G2.

FIELD OF THE INVENTION

The present invention relates to a hybridoma cell line 10G4 and amonoclonal antibody produced by the same that is capable of recognizingtotal aflatoxins.

BACKGROUND OF THE INVENTION

Aflatoxins, natural compounds toxic to human beings and animals, are thesecondary metabolites secreted by Aspergillus flavus and Aspergillusparasiticus. A variety of aflatoxins exist in the world and more than 20kinds have been identified. Aflatoxins are characterized by widepollution, strong toxicity and severe harm etc. Therefore, at least 70countries in the world have established maximum residue limit withregard to aflatoxins contained in agriculture products and foods. Manycountries have even established maximum residue limit with regard to thetotal amount of those main aflatoxins such as B1, B2, G1 and G2, so thetotal aflatoxin analysis is very important.

The current aflatoxin assay methods include thin layer chromatography(TLC), precision instrumental analysis and immunoassay. The TLC methodis the most popular for testing aflatoxins since it can be performed ingeneral labs and no special instruments are required. However, a lot ofreagents are needed for the TLC assay, the operation is complicated, andother components are liable to interfere with the test result, causingpoor accuracy, uncertain dosing and harm to operators and the ambientenvironment, which makes it unsuitable for quick on-site inspection. Theprecision instrumental analysis includes fluorescence spectrophotometryand high performance liquid chromatography (HPLC) characterized by highsensitivity and accuracy. However it is also unsuitable for quickinspection considering the expensive instruments, high purityrequirement for aflatoxin samples, complicated and time consuming samplepre-processing and strict testing environment requirements. Theimmunoassay technology developed in recent years can help to avoid theshortcomings of the former two methods since it is characterized by goodspecificity, high sensitivity, simple pre-processing, low cost, lessharm to operators and the ambient environment, and is suitable foron-site and batch inspection etc.

The immunoassy is adopted for qualitative and quantitative detection ofultra-micro residues based on the specific reaction of antigens andantibodies as well as the biological, physical or chemical magnificationof antigen (or antibody) markers. For any immunoassay techniques withrespect to total aflatoxin analysis, the antibody against totalaflatoxins B1, B2, G1 and G2 are required. In fact, many reportsregarding development of antibodies for anti-aflatoxins have beenpublished worldwide. Further, all-purpose antibodies (polyclonalantibodies) against aflatoxins have also been reported. Moreover, somescientists have established total aflatoxin assay for aflatoxins basedon the all-purpose anti-aflatoxin antibody. The all-purposeanti-aflatoxin antibody mainly features a strong, specific bindingreaction with different aflatoxins and can be used for establishingimmunoassay for any of the different aflatoxins. While the antibodiesagainst total aflatoxin B1, B2, G1 and G2 feature not only strongspecific binding reactions with different aflatoxins, but also,particularly, stronger sensitivity consistency with regard to theimmunoassay for each aflatoxin. The all-purpose antibody mentioned incurrent reports published both domestically and abroad foranti-aflatoxins shows high versatility, but as for each separateaflatoxin, the sensitivity consistency of the assay is poor. Thus, theall-purpose antibody mentioned in these reports is not suitable forestablishing the total aflatoxin assay for aflatoxins B1, B2, G1 and G2.Even if method of total aflatoxin assay is established based on thoseall-purpose antibody, the quantitative accuracy will be poor. Therefore,the development of antibodies against total aflatoxin B1, B2, G1 and G2is very important for quick quantitative immunoassay of the total amountof aflatoxin B1, B2, G1 and G2.

SUMMARY OF THE INVENTION

In one aspect, the present application is directed to a hybridoma cellline 10G4 and a monoclonal antibody produced by the hybridoma cell line10G4. The monoclonal antibody specifically recognizes total aflatoxinB1, B2, G1 and G2.

In one embodiment, the hybridoma cell line 10G4 was deposited at theChina Center for Type Culture Collection (CCTCC), Wuhan University,Wuhan, China on Jul. 13, 2010 with the accession number of CCTCC No.C201016. The scientific name of the hybridoma cell line 10G4 is “Mousehybridoma 10G4.” The hybridoma cell line 10G4 includes gene sequence asshown in SEQ ID NO.1 of the Sequence Listing that corresponding to theheavy chain variable (VH) region of the produced monoclonal antibody.The hybridoma cell line 10G4 also includes the gene sequence of as shownin SEQ ID NO.2 of the Sequence Listing that corresponding to the lightchain variable (VL) region of the produced monoclonal antibody. Theproduced monoclonal antibody is against total aflatoxin B1, B2, G1 andG2.

In another embodiment, the monoclonal antibody against total aflatoxinB1, B2, G1 and G2 is secreted by the hybridoma cell line 10G4 depositedin the China Center for Type Culture Collection (CCTCC) with theaccession number of CCTCC NO. C201016. VH region of the monoclonalantibody contains the amino acid sequence as shown in SEQ ID NO.3 of theSequence Listing and VL region of the monoclonal antibody contains theamino acid sequence as shown in SEQ ID NO: 4 of the Sequence Listing.

In certain embodiments, the hybridoma cell line 10G4 is obtained througha two-step selection method detailed as follows. The BALB/c mouse isimmunized with complete aflatoxin antigen AFB1-BSA 4-6 times, and thenis booster immunized the last time using twice the immunizing dose ofthe previous immunization. Next, cell fusion is conducted after threedays. The fusion cells are selected by a two-step enzyme-linkedimmunosorbent assay (ELISA). Step one, selecting positive wells whichare against aflatoxin but not against carrier protein BSA throughindirect ELISA. Step two, testing culture solution of the positive wellsselected via step one through indirect competitive inhibition ELISA. Instep two, aflatoxin G2 can be used as the competition source—because theall-purpose antibody against aflatoxins reported worldwide shows thelowest cross reacting rate against aflatoxin G2 (<50%). The cloning iscarried out through a limiting dilution method after the wells with highlight absorption value and sensitivity are selected. The same two-stepselection is performed after about 10 days from cloning. The cloningprocess is repeated 2-3 times, and the hybridoma cell line 10G4 isfinally obtained.

The monoclonal antibody against total aflatoxin B1, B2, G1 and G2 can beused for measurement of total aflatoxin B1, B2, G1 and G2.

In certain embodiments, monoclonal antibody against total aflatoxin B1,B2, G1 and G2 are prepared as follows. A BALB/c mouse is pretreated byFreund's incomplete adjuvant. Then the treated BALB/c mouse is injectedwith the obtained hybridoma cell line 10G4. The ascites of the mouse iscollected, and the monoclonal antibody against total aflatoxin B1, B2,G1 and G2 are obtained after purification.

In one embodiment, the purification is carried out through the caprylicacid-ammonium sulfate (CA-AS) precipitation method detailed as follows.The mouse ascites is filtered with a piece of double-layer filter paper.The filtrate is centrifuged at 12000 r/min under 4° C. for more than 15minutes (min), and the supernatant is pipetted. The supernatant of theascites is mixed with four times volume of acetate buffer solution, andn-Caprylic acid is added slowly while stirring. The volume of n-Caprylicacid needed for each milliliter (mL) of ascites is about 30˜35 μL. Thesolution is mixed at room temperature for 30˜60 min and then kept at 4°C. for more than 2 hours (h). Then the above solution is centrifuged at12000 r/min under 4° C. for more than 30 min. The precipitate isdiscarded and the obtained supernatant is filtered with a piece ofdouble-layer filter paper. 0.1 mol/L (M), pH 7.4 phosphate buffersolution ( 1/10 of the volume of the filtrate) is added to the filtrate,and the pH value of the mixture is adjusted to 7.4 with 2 M sodiumhydroxide solution and precooled to about 4° C. Ammonium sulfate isadded slowly to the mixture until the final concentration of ammoniumsulfate reached 0.277 g/mL. The solution is kept for more than 2 h at 4°C. and then centrifuged at 12000 r/min under 4° C. for more than 30minutes. The supernatant is discarded and the obtained precipitate isre-suspended with 0.01 M phosphate buffer solution that is 1/10 of thevolume of the original ascites. The re-suspended solution is put into adialysis bag for dialysis against pure water. The completely dialyzedprotein solution is frozen at a −70° C. freezer, and then lyophilizedwith a lyophilizer. The lyophilized powder is collected, which is thepurified monoclonal antibody against total aflatoxin B1, B2, G1 and G2.The purified antibody can then be stored at −20° C. for future use.

In the above embodiment, the acetate buffer solution is prepared bymixing 0.29 gram (g) sodium acetate, 0.141 mL acetic acid, and water toa total volume of 100 mL. The 0.1 M phosphate buffer solution isprepared by mixing 0.8 g sodium chloride, 0.29 g sodium hydrogenphosphate dodecahydrate, 0.02 g potassium chloride and 0.02 g potassiumdihydrogen phosphate and water to a total volume of 100 mL.

The present invention, among other things, has the following advantages:

(1) The hybridoma cell line 10G4 can be used to produce the monoclonalantibody against total aflatoxin B1, B2, G1 and G2. The titer of the10G4 mouse ascites antibody measured through non-competitive ELISA canreach up to 5.12×10⁵.

(2) The monoclonal antibody against total aflatoxin B1, B2, G1 and G2can be used for better identification of aflatoxin B1, B2, G1 and G2with good consistency. The 50% inhibitory concentrations (IC₅₀) foraflatoxin B1, B2, G1 and G2 are 2.09 nanogram(ng)/mL, 2.23 ng/mL, 2.19ng/mL and 3.21 ng/mL respectively. The range of cross reacting rate foraflatoxin B1, B2, G1 and G2 is about 65.2%-100%.

(3) The monoclonal antibody against total aflatoxin B1, B2, G1 and G2can be used for quantitative measurement of total aflatoxin B1, B2, G1and G2.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Preparation of HybridomaCell Line 10G4

1. Animal Immunization

Six 6-week old BALB/c mice are immunized by the complete aflatoxin B1antigen AFB1-BSA. The first immunization: emulsifying the completeaflatoxin B1 antigen and the equivalent amount of Freund's completeadjuvant, then multiple-point injecting the mice with the emulsion atthe nape subcutaneously. The second immunization is performed after 4weeks: emulsifying the complete aflatoxin B1 antigen and the equivalentamount of Freund's incomplete adjuvant, then injecting the mice with theemulsion intraperitoneally. The third immunization is performed 4 weeksafter the second immunization, and the immunization method is the sameas the second immunization. The fourth immunization is performed 3 weeksafter the third immunization, and the immunization method is the same asthe second immunization, i.e., intraperitoneal injection. The dose usedfor the 4 immunization processes can be the same, i.e., 50 μg for eachmouse. For the first 3 immunizations, 8 days after each immunization,the blood is collected from the caudal vein, the serum is separated, andthe mouse serum titer is monitored through indirect ELISA. 8 days afterthe fourth immunization, the blood is collected from the caudal vein,the serum is separated, the mouse serum titer is monitored throughindirect ELISA method, and the serum sensitivity of the mice ismonitored through indirect competitive ELISA. The mouse with relativelyhigher tilter and serum sensitivity than the other mice is chosen toperform the last booster immunization. Dose of the booster immunizationis twice as those used in the previously immunization. The completeaflatoxin B1 antigen AFB1-BSA can be purchased from Sigma-Aldrich.

2. Cell Fusion

Three days after the final booster immunization, cell fusion isperformed according to the common method using 50% (percentage byweight) polyethylene glycol (PEG, molecular weight: 1450) as fusionagent. The cell fusion is detailed as follows. The mouse is killed underaseptic conditions. Splenocytes are separated and mixed with mousemyeloma cells SP2/0 at a 5:1 ratio. The mixed cells are rinsed withRPMI-1640 media, and fused using the 50% PEG for 1 min. Then thecontainer is filled with RPMI-1640 media. The solution is centrifugedand the supernatant is discarded. The fused cells of the splenocytes andmouse myeloma cells are re-suspended with 72 mL RPMI-1640 media. There-suspended cells is pipetted into a 96-well cell culture plate (2drops/well) and then cultured in a carbon dioxide incubator at 30° C.The RPMI-1640 media contains 20% (percentage by volume) of fetal calfserum, 2% (percentage by weight) of growth factor and 1% (percentage byweight) of hypoxanthine-aminopterin-thymidine (HAT). The above SP2/0 canbe purchased from Shanghai Fankel Biological Technology Co., Ltd. TheRPMI-1640 media can be purchase from Hyclone company. The HAT can bepurchased from Sigma-Aldrich.

3. Selection and Cloning of Hybridoma Cells

After the cell fusion is kept for about 12 days, the cell colony growsto fill ½ of the well bottom and the culture solution becomes yellow,and antibody can be analyzed. The culture wells with hybridomas areselected through ELISA, which is performed in two steps: selectingpositive wells which are against aflatoxin B1 rather than the carrierprotein BSA through indirect ELISA; and testing the selected positivewells via step one through indirect competitive inhibition ELISA. Theaflatoxin G2 can be used as competition antigen. Wells with high lightabsorption value and sensitivity (high light absorption value means thatthose wells with the competition source being 0, i.e., the positivecontrol wells, have high measured values; high sensitivity means thatthose wells that has 50% inhibition rate, i.e., the IC₅₀ value, is low)are selected. Cloning is performed through limiting dilution method.After about 10 days from the cloning, the same two-step selection methodis conducted. Repeat the selection and cloning 2-3 times, and thehybridoma cell line 10G4 is then obtained.

Example 2

Variable region sequencing for hybridoma cell line 10G4 that producedmonoclonal antibody against total aflatoxin B1, B2, G1 and G2.

(1) Extracting total RNA: the total RNA extraction kit of TiangenBiotech (Beijing) Co., Ltd is used and the total RNA of the hybridomacell line 10G4 is extracted according to protocol of the kit.

(2) Synthesizing cDNA: the first chain of cDNA is synthesized by takingthe total RNA obtained via Step (1) as template, the oligo(dT)₁₅ asprimer, and performing the reverse transcription according to theSuperScriptTM-2 II Reverse Transcription protocol. The Primeroligo(dT)_(is) is purchased from Invitrogen.

(3) Cloning variable region genes through PCR: a primer is designedaccording to the conserved sites of the mouse antibody gene sequence inGENEBANK, and the VL/VH region are amplified by taking cDNA as template.PCR procedure: 94° C. 30 seconds, 55° C. 1 min, 72° C. 1 min, 30 cyclesof amplification, the last operation at 72° C. is prolonged to 10 min.The PCR product is separated by 1% (percentage by weight) agarose gelelectrophoresis, and the DNA segments are purified. The purified DNAsegment is inserted to the carrier pMD18-T and the inserted carrierpMD18-T is used to transform the E. coli DH5α competent cells. Positivecolonies are selected and sent to Shanghai Sunny Biotechnology Co., Ltdfor DNA sequencing. Sequences of the VH region primers are: 5′-AGG TSMARC TGC AGS AGT CWG G-3′ (22 mer) and 5′-TGA GGA GAC GGT GAC CGT GGT CCCTTG GCC CC-3′ (32 mer), where S, M, R and W are degenerate bases, M=A/C,R=A/G, S=C/G and W=A/T. Sequences of the VL region primers are: 5′-GACATT GAG CTC ACC CAG CTT GGT GCC-3′ (24 mer) and 5′-CCG TTT CAG CTC CAGCTT GGT CCC-3′(24 mer).

Obtained gene sequences: the length of the gene sequence encoding the VHregion is 356 bp, see SEQ ID NO: 1 for the detailed sequence. Accordingto the obtained gene sequence, the VH region encoded by this genesequence has 118 amino acids, see SEQ ID NO: 3 for the detailedsequence. The length of the gene sequence encoding the VL region is 332bp, see SEQ ID NO: 2 for the detailed sequence. According to theobtained gene sequence, the VL region encoded by this gene sequence has110 amino acids, see SEQ ID NO:4 for the detailed sequence.

Example 3

Preparation, purification, subtype and characteristic assay ofmonoclonal antibody against total aflatoxin B1, B2, G1 and G2.

A BALB/c mouse is treated with Freund's incomplete adjuvant, and then isinjected with the hybridoma cell line 10G4 obtained from Example 2 whichcan produce monoclonal antibody against total aflatoxin B1, B2, G1 andG2. The ascites of this mouse is collected and antibody is purifiedthrough the caprylic acid-ammonium sulfate (CA-AS) precipitation methoddetailed as follows. The mouse ascites is filtered with a piece ofdouble-layer filter paper. The filtrate is centrifuged at 12000 r/minunder 4° C. for more than 15 min. The supernatant is pipetted. Thesupernatant of the ascites is mixed with acetate buffer solution (4times as much as the former). N-caprylic acid is added slowly whilestirring. The volume of n-caprylic acid needed for each mL of ascites isabout 33 μL. The mixture is kept at room temperature for 30˜60 min andthen stored at 4° C. for more than 2 h. The mixture is centrifuged at12000 r/min under 4° C. for more than 30 min. The precipitate isdiscarded and the obtained supernatant is filtered with a piece ofdouble-layer filter paper. Phosphate buffer solution ( 1/10 of thevolume of the filtrate, mol concentration: 0.1 mol/L, pH value: 7.4) isadded to the filtrate. The pH value of this mixture is adjusted to 7.4using 2 mol/L sodium hydroxide solution and the pH adjusted mixture isprecooled to 4° C. Ammonium sulfate is added slowly until the finalconcentration of ammonium sulfate reaches 0.277 g/mL in the solution.The solution is kept for more than 2 h at 4° C. and then centrifuged at12000 r/min under 4° C. for more than 30 min. The supernatant is removedand the obtained precipitate is re-suspended with 0.01 mol/L phosphatebuffer solution that is 1/10 of the volume of the original ascites. There-suspended solution is put into a dialysis bag for dialysis againstpure water. The completely dialyzed protein solution is frozen at −70°C. in a freezer, and then lyophilized with a lyophilizer. Thelyophilized powder, i.e., the purified monoclonal antibody against totalaflatoxin B1, B2, G1 and G2, is collected. The purified antibody isstored in a freezer at −20° C. for future use. The acetate buffersolution is prepared by adding 0.29 g of sodium acetate and 0.141 mL ofacetic acid into water to a total volume of 100 mL. The 0.1 mol/Lphosphate buffer solution is prepared by adding 0.8 g of sodiumchloride, 0.29 g of sodium hydrogen phosphate dodecahydrate, 0.02 g ofpotassium chloride and 0.02 g of potassium dihydrogen phosphate intowater to a total volume of 100 mL.

The subtype of the monoclonal antibody against total aflatoxin B1, B2,G1 and G2, which is secreted by the hybridoma cell line 10G4, isdetermined as IgG1 using subtype kit available on the market.

The titer of the mouse ascites antibodies of the 10G4 mouse isdetermined by non-competitive ELISA and the titer can reach up to5.12×10⁵. That is, the test result is positive even after the mouseascites antibody has a 5.12×10⁵-fold dilution. The sensitivity of theantibody against total aflatoxin B1, B2, G1 and G2, is determinedaccording to indirect competitive inhibition ELISA. The 50% inhibitoryconcentrations (IC₅₀) are 2.09 ng/mL, 2.23 ng/mL, 2.19 ng/mL and 3.21ng/mL respectively. The range of cross reacting rate for aflatoxin B1,B2, G1 and G2 is 65.2%-100%.

Example 4 Application of Monoclonal Antibody 10G4 against TotalAflatoxin B1, B2, G1 and G2

4 standard curves are obtained according to Example 3 in respect to theresult against aflatoxin B1, B2, G1 and G2. A new quantitative analysiscurve for analyzing total aflatoxin of B1, B2, G1 and G2 is constructedby an averaging process of the 4 standard curves. The new curve is usedto test total aflatoxin B1, B2, G1 and G2 of five sets of peanutsamples. At the same time, a comparison analysis based on standard highperformance GB/T 18979-2003 liquid chromatogram method is performed. Theresults from the above two methods shown high consistency. The detailedresults are as follows:

Sample 1: total aflatoxin determined based on antibody 10G4 ELISA is0.23 ng/mL; total aflatoxin determined by the high performance GB/T18979-2003 liquid chromatogram method is 0.2 ng/mL;

Sample 2: total aflatoxin determined based on antibody 10G4 ELISA is1.47 ng/mL; total aflatoxin determined by the high performance GB/T18979-2003 liquid chromatogram method is 1.2 ng/mL;

Sample 3: total aflatoxin determined based on antibody 10G4 ELISA is0.88 ng/mL; total aflatoxin determined by the high performance GB/T18979-2003 liquid chromatogram method is 1.1 ng/mL;

Sample 4: total aflatoxin determined based on antibody 10G4 ELISA isnegative; total aflatoxin determined by the high performance GB/T18979-2003 liquid chromatogram method is negative;

Sample 5: total aflatoxin determined based on antibody 10G4 ELISA is0.92 ng/mL; total aflatoxin determined by the high performance GB/T18979-2003 liquid chromatogram method is 0.8 ng/mL.

What is claimed is:
 1. A hybridoma cell line 10G4 deposited at the ChinaCenter for Type Culture Collection (CCTCC) with the CCTCC accessionnumber of C201016.
 2. A monoclonal antibody against total aflatoxin B1,B2, G1 and G2, wherein the monoclonal antibody is produced by hybridomacell line 10G4 that is deposited at the China Center for Type CultureCollection (CCTCC) with the CCTCC accession number of C201016.
 3. Anapplication of the monoclonal antibody of claim 2 to measure totalaflatoxin B1, B2, G1 and G2.
 4. A method for producing the monoclonalantibody of claim 2, comprising: treating a BALB/c mouse by Freund'sincomplete adjuvant; injecting the treated BALB/c mouse with hybridomacell line 10G4; collecting ascites of the BALA/c mouse; and purifyingthe monoclonal antibody of claim 2 from the ascites.